The research program is concerned with the analysis, synthesis, structure-function relationships of peptide and protein hormones, and proteomics. Development of one-platform tandem mass spectrometric analysis for the identification of serine and threonine phosphorylation and glycosylation sites in protein, screening of angiotensin II (AngII)-mediated changes in the protein kinase expression and phosphorylation site levels in cells, and structure-function studies of an anti-HIV peptide from human lysozyme were accomplishments of the past year. These include (1) Glycosylation of proteins occurs essentially in one of two forms, Asn-(N)-linked and Ser- or Thr (O)-linked. While the N-linked glycosylation site can be predicted by its occurrence on Asn in Asn-X-Ser /Thr sequence and identified by protein sequence analysis as Asp after a specific glycosidase cleavage, similar principles are not applicable for O-glycosylation sites. In addition, tandem mass spectrometric analyses (MS/MS) of a glycopeptide is impractical due to heterogeneities and multiple chain lengths of the oligosaccharide moieties attached to the peptide, generating highly complex spectra that are impossible to interpret. Therefore, developing an effective O-linked glycoprotein analysis method is necessary in order to tackle many problems associated with carbohydrate-mediated biological functions. Previously, methodologies based on beta-elimination and Michael addition have been successfully developed under this program for the identification of P-Ser and P-Thr sites in peptides by MS/MS. In particular, an effective modification of phosphopeptide was achieved under a mild base-catalyzed beta-elimination using 20 mM Ba-hydroxide and addition of propanethiol at 0.5M in 2M n-propanol that forms S-propylcysteine and beta-methyl-S-propylcysteine from P-Ser and P-Thr, respectively. Although Ba-hydroxide has long been considered ineffective for the beta-elimination of O-glycosidic Ser and ?Thr because the carbohydrate moieties do not form coordinate with barium ion. However, beta-elimination of O-glycosidic-Ser or ?Thr is known to occur readily at a lower sodium hydroxide concentration than that of P-Ser or P-Thr. Indeed, tests of synthetic O-linked N-acetylgalactosamidic-Ser glycopeptide (KMSTLgSYR, g denotes the monosaccharide residue) under the condition for phosphopeptides detected the formation of S-propylcysteine at the O-glycosidic site by ESI ion trap MS/MS. Conditions of 50-100 mM Ba-hydroxide, 25% DMF, 20% ETOH, and 1M propanethiol, incubated for 24 hours at room temperature were developed and applied to modify two natural glycopeptides (Sg?TVATLEDpSPE and Pg?TSg?TPTg?TEAVE, g? denotes complex oligosaccharides) purified from a trypsin and Glu-C digest of glycomacropeptide from bovine kappa-casein. Both phosphorylation and O-glycosylation sites in these peptides were elucidated by ESI ion trap MS/MS. (2) In addition to blood pressure regulation, aldosterone secretion, and sodium balance, AngII has potent mitogenic and pro-inflammatory activities, which influence multiple cellular responses involving in vascular inflammation and structural remodeling. Although the primary action of AngII is mediated by the Gq-coupled AT1 receptor and its inositol phosphate/Ca+2 mediated signaling pathways, protein kinases and phosphoproteins have pivotal roles in controlling responses. In search of novel protein kinases with such a role, we took advantage of the availability of specific antibodies against a wide variety of protein kinases, and analyzed expression patterns of protein kinases by the western blot analysis before and after AngII stimulation in human adrenocortical carcinoma cells (H295R). The results indicated that AngII stimulation increased levels of casein kinase 2 (35kD, 26%); elongation factor-2 protein-serine kinase (105kD, 134%); ERK1 (40kD, 20%); IKKbeta (87kD, 63%); AKT/PKBalpha (57kD, 77%); PKC-micro (120kD, 40%); Moloney sarcoma oncogene-encoded protein-serine kinase (34kD, 52%); DAPK (156kD, 122%); FAK (123kD, 55%) and JAK1 (118kD, 65%). In contrast, AngII stimulation of H295R also resulted in decreased levels of other kinases. Decreases were seen in Cdk 5 (28kD, 33%); Osaka thyroid oncogene protein-serine kinase (53kD, 25%); GSK3-alpha (44kD, 45%); PKCalpha (75kD, 47%); ribosomal S6 kinase 1 (84kD, 21%); CaM kinase 1 (38kD, 32%); IKK-alpha (78kD, 21%); Yes-related protein-tyrosine kinase (45 KD, 51%); Raf1 proto-oncogene-encoded protein-serine kinase (69kD & 73kD, 46% & 41%); cGMP-dependent protein kinase (70kD, 20%) and ZIP kinase (45kD, 46%). We also utilized anti-phospho-site antibodies to analyze the level of phosphorylation at specific sites in protein kinases in AngII-stimulated H295R cells. Results indicate increases of 90kD ribosomal S6 kinases at S380/386,139% and T573/577, 82%; AMP-activated protein kinase-alpha (T174/172, 73%); ERK1 (T202/Y204, 63%); GSK3beta (S9 38%); MEK1/2 (S212/221, 22%); p38alpha MAPK (T180/Y182, 61%); p70 S6 kinase (T421/424, 55%); Raf1 (60kD, S259, 181%); Raf1 (70kD, S259, 27%). Decrease of the phosphorylation level was observed in Cdk1 (T161/160, 61%); Cdk1 (Y15, 17%) and GSK3alpha (S21, 43%). These results indicate increases of the expression of p38MAPK and ERK as well as phosphorylation of their specific sites in agreement with previous studies. The increased levels of PKB/Akt and GSK3beta phosphorylation at Ser9 are noteworthy. GSK3beta is known to be phosphorylated at Ser9 by PKB/Akt after stimulation by a wide variety of agents. As a consequence, the activity of GSK3beta is attenuated to negatively regulate downstream signaling pathway involving phosphorylation of several transcription factors. PKB/Akt is also a major kinase downstream from phosphoinosito-3-kinase (PI3K). Therefore, many stimuli that activate PI3K may also inhibit GSK3beta through PKB/Akt phosphorylation. Our results establish for the first time a linkage of AngII stimulation and GSK3beta phosphorylation. Although negative regulation of GSK3beta causes cardiac hypertrophy, there is no report of AngII-induced GSK3 effect in either cardiac or adrenal cells. It is conceivable that the cardiac hypertrophic effect of AngII could be mediated through the Gq-coupled AT1 and the PI3K-PKB/Akt to GSK3beta pathway. (3) Lysozyme was originally described for its effects against Gram-positive bacteria. This bactericidal property is due to its muramidase activity. However, reduced and/or partially unfolded lysozyme still exerts broad spectrum antimicrobial action against both Gram-negative and Gram-positive bacteria, independent of muramidase activity. Moreover, lysozyme is widespread in nature and plays critical roles in host defense, both in the control of microbial infection and in the modulation of host immunity. Our previous report has indicated that lysozyme accounts for the anti-HIV activity associated with the beta-core fraction of human chorionic gonadotropin. In search of the active sites for anti-HIV activities, peptide fragmentation of human lysozyme and activity mapping were conducted. Two fragments denoted as HL18 and HL9, were identified which possess full anti-HIV activity comparable to the intact molecule. Structure-function studies based on circular dichromic measurements, energy minimization calculations of various HL9 analogues as well as their bacteria lysis activity and anti-HIV activity in cells have revealed that the anti-HIV activity of peptide HL9 cannot be fully explained by its basicity (pI 12.3) or its secondary structure alone. Other peptides with identical pIs and alpha-helical structure, such as the HL9 scrambled sequence and the HL9 R to K mutants, do not have anti-HIV activity. Unique amino acid sequence, specific structural features, and folding topologies must be required for anti-HIV activity.